Fesselin, a Synaptopodin-like Protein, Stimulates Actin Nucleation and Polymerization

Abstract

Fesselin is a proline-rich actin binding protein that has recently been isolated from smooth muscle [Leinweber, B. D., Fredricksen, R. S., Hoffman, D. R., and Chalovich, J. M. (1999) J. Muscle Res. Cell Motil. 20,[539][540][541][542][543][544][545]. Fesselin is similar to synaptopodin [Mundel, P., Heid, H. W., Mundel, T. M., Krüger, M., Reiser, J., and Kriz, W. (1997) J. Cell Biol. 139, 193-204] in terms of its size, isoelectric point, and sequence although synaptopodin is not present in smooth muscle. The function of fesselin is unknown. Evidence is presented here that fesselin accelerates the polymerization of actin. Fesselin was effective on actin isolated from either smooth or skeletal muscle at low ionic strength and in the presence of 100 mM KCl. At low ionic strength, fesselin decreased the time for 50% polymerization to about 1% of that in the absence of fesselin. The lag phase characteristic of the slow nucleation process of polymerization was eliminated as the fesselin concentration was increased from very low levels. Fesselin did not alter the critical concentration for actin but did increase the rate of elongation by ≈3-fold. The increase in elongation rate constant is insufficient to account for the total increase in polymerization rate. It is likely that fesselin stabilizes the formation of actin nuclei. Time courses of actin polymerization at varied fesselin concentrations and varied actin concentrations were simulated by increasing the rate of nucleation and both the forward and reverse rate constants for elongation.Fesselin is an actin binding protein that has recently been isolated from turkey gizzard muscle (1). Fesselin is noteworthy because of its potent ability to bundle actin and because of its similarity to synaptopodin, a protein found in telencephalic dendrites and renal podocytes but not in smooth muscle (2). Fesselin has in common with synaptopodin a similar pI (9.3), a similar mobility on SDS gels (103 and 79 kDa for fesselin and 110 kDa for synaptopodin), a high proline content, and regions of sequence homology. Antibodies directed against synaptopodin decorate actin filaments in cells forming a punctate pattern (2); this pattern is lost following depolymerization of actin with cytochalasin B. Preliminary studies with fesselin also indicate that fesselin co-localizes with α-actinin on actin filaments (3). The functions of fesselin and synaptopodin are unknown.The effect of fesselin on actin polymerization was examined because of its ability to bind to and bundle actin filaments and its high isoelectric point. The isoelectric point was a consideration because cationic substances tend to polymerize actin (4,5). Actin binding and polymerizing proteins are often basic (6), and charge neutralization may be a factor in the function of these proteins (5).The present study provides evidence that fesselin increases the rate of actin polymerization. This was seen by increases in the apparent rates of increase of both pyrene fluorescence and † Funded by NIH Grant AR35216 and a Facult...

Regulation of Smooth Muscle Dystrophin and Synaptopodin 2 Expression by Actin Polymerization and Vascular Injury

“…Both fesselin and myopodin have been demonstrated to bind to actin filaments and participate in actin polymerization by formation of actin bundles. 25,39 We found that Synpo2 mRNA and protein were abundant in differentiated vascular smooth muscle and dramatically reduced in proliferating SMCs. Interestingly, the expression of Synpo2 was also significantly reduced in human arteries after balloon dilation, indicating a potential role in restenosis after angioplasty.…”

“…25 To determine whether knockdown of Synpo2 is sufficient to cause actin depolymerization and loss of smooth muscle marker expression, we transfected cultured SMCs with Synpo2 GapmeRs. A combination of 4 different GapmeRs was used to achieve maximal knockdown.…”

Synaptopodin family of natively unfolded, actin binding proteins: physical properties and potential biological functions

“…Synaptopodin 2 from avian gizzards stimulates the rate of polymerization of both rabbit skeletal and turkey gizzard actin (Beall and Chalovich 2001). The polymerization rate increased with increasing avian synaptopodin 2 concentrations; at low ionic strength conditions 75 nM avian synaptopodin 2 increased the rate of actin polymerization by 33-fold.…”

Actin polymerization is stimulated by actin cross-linking protein palladin

“…Briefly, G-actin was polymerized at a high concentration (20 μM) by adding MKEI polymerization buffer, the mixture was incubated at room temperature for 1 hour and subsequently vortexed for 30 s to use fragments as a seeds for elongation. The concentration of vortexed F-actin seeds relative to the total actin concentration was below 5% [27]. The spontaneous barbed-end assembly was performed using 5 μM, 5% pyrene actin, 20 μM Palld-Ig3 and 0.5 μM F-actin seeds.…”